This proposal aims to test the hypothesis that type II afferents serve as cochlear nociceptors. Taking cues from the human complaint of hyperacusis after hearing loss, we will examine the structure and function of type II afferents in normal and post?trauma cochleas. The working hypothesis is that painful hyperacusis, noxacusis, includes hyperactivity of type II afferents, by analogy to hyperalgesia of somatic nociceptive C?fibers. Thus we will examine type II structure and function in normal and post?trauma cochleas of rats and mice. In parallel we will investigate the properties of surviving outer hair cells in post? trauma cochleas. Our methods include: ex vivo electrophysiology, light and electron microscopy, utilization of optogenetic and chemogenetic tools,and validation and quantification of mouse models in which type II specific bio?markers are expressed. A necessary first step is to extend our ex vivo experimental approach to older cochleas so that changes wrought by acoustic trauma can be compared to the normal condition. We will compare damaging sound, ototoxic antibiotics and genetically encoded biotoxins to produce experimentally tractable effects on tissue for ex vivo experiments. The properties and synaptic connections of type II afferents and outer hair cells will be examined in the excised cochlear tissue of these animals. We will continue to explore type II specific genetic mouse models. Genetically?encoded reporter proteins, voltage? and calcium?sensitive indicators, biotoxins, and opto? and chemo?genetic modulators have become highly informative tools in neurobiology generally and for the inner ear specifically. Our ongoing work has characterized one mouse line, tyrosine hydroxylase promoter driven Cre?recombinase expression. Three other candidate type II specific Cre lines will be validated and quantified. With such transgenic models it becomes possible to study innervation patterns by expression of fluorescent reporter proteins, and to activate, eliminate, or modulate type II activity for anatomical and physiological studies. Cre?dependent expression of genetically?modified G?protein?coupled receptors (DREADDS) will provide mice in which type II activity can be increased or decreased by injection of a novel synthetic ligand, depending on the specific construct. Varying combinations of systemic and round window drug delivery will be employed to increase the specificity of experimental manipulations. The over?arching goal of this program of experiments is to complete the description of type II afferents, a still?unresolved component of cochlear innervation. The working hypothesis is that these serve as cochlear nociceptors. If correct these are a likely neurobiological substrate for noxacusis (painful hyperacusis). By defining the basic cellular and molecular mechanisms of type II function and plasticity, future therapeutic targets can be identified to ameliorate or prevent noxacusis.